1. Field of the Invention
This invention relates to the field of tissue engineering, and more particularly to a system and method for producing a connective tissue construct, such as a tendon construct, in vitro.
2. Background Art
There are approximately 33 million musculoskeletal injuries each year in the United States. The associated soft tissues, which include tendons, comprise almost 50% of these injuries. In some cases, the tendon is damaged beyond repair, and partial or whole replacement of the tendon is necessary. The ideal replacement would be autologous tendon, but transplantation is limited by the availability of viable autograft tissue. As a result, clinical practice has turned to the use of synthetic materials (see Goldstein et al., Journal of Bone and Joint Surgery—American Volume, 71A, p. 1183, 1989), where current synthetic replacements include DACRON® grafts, carbon fibers, and silastic sheets. Unfortunately, these materials are unable to adequately restore function for the long term due to their inherent mechanical incompatibility with the in vivo environment as well as their tendency to degrade (see Iannace et al., Biomaterials, 16, p. 675, 1995).
Tendons are densely packed connective tissues that transmit the forces between muscle and bone. They are stiff in tension, yet flexible enough to conform to their anatomical environment. The material properties of tendon tissue can be attributed to the parallel fibrils of collagen which make up approximately 75% of the dry weight of adult tendons. In the resting state, the fibrils display a periodic wavy pattern, defined as the crimp. As a tendon is stretched, the crimped collagen fibrils begin to straighten out and may cause the tendon to become stiffer with increasing mechanical strain. Tendons have a low cell density, around 20% of the tissue volume, but fibroblasts are integral in the development and maintenance of the tissue. The distinct spatial orientation of tendon fibroblasts is associated with the organization of collagen fibers into the hierarchical tendon structure.
Because of its relatively avascular nature, tendon is a prime candidate for engineered tissue replacement. Previous attempts have been made to create biologically based tendons in vitro, but these have met with limited success due to the difficulty in creating an in vitro tissue, or “construct”, that is both mechanically and biologically compatible with the in vivo environment (see Butler and Awad, Clinical Orthopedics and Related Research, 367, p. S324, 1999; Goldstein et al., Journal of Bone and Joint Surgery—American Volume, 71A, p. 1183, 1989; Torres et al., Biomaterials, 21, p. 1607, 2000; Cao et al., Plastic and Reconstructive Surgery, 110, p. 1280, 2002; Koob and Hernandez, Biomaterials, 23; p. 203, 2002).
Mechanical difficulties can arise from the reliance on artificial scaffolds when attempting to engineer tendon. Type I collagen is the most widely used scaffold material since it was observed that fibroblasts will contract a collagen gel to form a tissue-like structure (see Bell et al., Proceedings of the National Academy of Sciences of the United States of America, 76, p. 1274, 1979). Collagen would appear to be the ideal foundation for an artificial tendon, but presently the mechanical properties of in vitro fibroblast-collagen constructs are inferior to those of native tissues (see Huang et al., Annals of Biomedical Engineering, 21, p. 289, 1993; Wakatsuki et al., Biophysical Journal, 79, p. 2353, 2000; Seliktar et al., Annals of Biomedical Engineering, 28, p. 351, 2000; Brown et al., Journal of Cellular Physiology, 175, p. 323, 1998; Cacou et al., Medical Engineering & Physics, 22, p. 327, 2000).
An explanation for this discrepancy is that gelled collagen is generally disorganized and only forms fibrils of physiological thickness under stringent conditions (see Holmes et al., Journal of Biological Macromolecules, 8, p. 161, 1986). Furthermore, native tendons possess an extracellular matrix (ECM) composed of many proteins, glycosaminoglycans, and proteoglycans which control the assembly of the collagen fibril, the load bearing unit, and contribute to the formation of the tissue hierarchy. Fibroblasts rely on cell-matrix signaling pathways during development to properly assemble the fibrils and maintain form and function after maturation. Koob and Hernandez created a mechanically relevant construct by cross-linking extruded collagen fibers with NGDA, a plant derived anti-oxidant, for which only ultimate strengths were reported and not the entire elastic response (see Koob and Hernandez, Biomaterials, 23; p. 203, 2002). Goldstein et al. used the same idea of creating a fiber composite to create artificial prostheses, but relied upon cross-linking methods that were cytotoxic and/or non-biodegradable (see Goldstein et al., Journal of Bone and Joint Surgery—American Volume, 71A, p. 1183, 1989).
Accordingly, a need exists for a tendon construct that incorporates as many of the native properties of tendon as possible in order to sufficiently restore function.